When working with man-made materials and naturally occurring substances, engineers and scientists are accustomed to observing and measuring physical characteristics and behavioral phenomena such as size, shape, dimensions, transformation, motion, and other cause and effect relationships on the micrometer (μm) scale. For example, in the electronics and semiconductor industry, the dimensions of devices are measured in terms of microns. In biotechnology applications, most instruments and techniques used to observe physical characteristics and behavioral phenomena, including microscopes, optical imaging, and electron micrographs are also in the micrometer range.
As technology advances, the dimensions of devices, materials, and substances of interest continue to dramatically shrink in size. Significant research and development is underway in what is commonly known as nanotechnology, i.e., devices and materials that exist and operate in the range of 1 to 1000 nanometers (nm). It has been said that the ultimate refinement of realization and sensitivity is a single molecule. With nanotechnology, work is often done at the molecular level. Complex processes can take place in such a small space that the application become very portable. Propagation times and energy consumption are negligible.
Nanotechnology finds applications in characterizing and monitoring nanoscopic systems ranging from single molecules to nano-electro-mechanical and nanofluidic systems. Researchers continue to look for new applications of nanotechnology. The concept of realizing independently operating, man-made and engineered devices measured in terms of nanometers has become reality and will continue to progress. And, as the field of biotechnology advances, the need to observe, measure, manipulate, control, and test substances and elements at the molecular level is ever more present.
One of the behavioral phenomena that exist in the world of nanotechnology is motion. Many aspects of the nanoworld are continuously in motion. The nature of the motion is directly related to the physical characteristics and environment to which the nanoscale elements and structures are subjected. The ability to detect, observe, measure, and control such motion at the nanometer scale is important to the continuation of research and development of new products and design methods. Modern instrumentation and research techniques have difficulty with the accurate and reliable detection of motion, particularly rotational motion, in the nanometer range.
Attempts have been made to detect and measure rotational motion of small particles by observing changes in the orientation of the particle over time under a microscope. For example, the F1-ATPase enzyme has been observed to exhibit rotational motion along its axis by using fluorescence microscopy to visualize time-dependent changes in the orientation of fluorescently labeled actin filaments, a protein which is about 0.5 to 4 μm long, attached to the rotating shaft of the enzyme. Other research has measured the rotation of anisotropically patterned fluorescent polymer microspheres in the range of 2 to 4 .mu.m in diameter. The particles of interest possess sufficient anisotropy to allow its orientation to be seen under the microscope.
Alternatively, if a particle rotates about an axis that is not an axis of symmetry of the particle, then its rotation can be measured by tracking the centroid of the particle's image. The rotation of a 1 .mu.m polystyrene sphere attached to the shaft of the F1-ATPase molecule has been measured by detecting the displacement of the centroid of the sphere's image due to the slight eccentricity in the shaft's rotation.
At the nanoscale, the direct approach to detecting rotational motion using a light-based microscope is usually ineffective, since the diffraction limit of the light makes it difficult to resolve features, and hence determine the orientation, of nanoscopic objects. That is, the magnitude of the change in position of the object is less than the diffraction limit of the light used to measure it.
The single-molecule fluorescence polarization spectroscopy and the centroid-tracking method have limitations in that the signal emitted by a single fluorophore is weak. Fluorescence polarization spectroscopy requires a sensitive optical detection system. In addition, the probe is susceptible to photo-bleaching. Finally, the intensity of emission of a single fluorophore fluctuates. Single-molecule fluorescence polarization spectroscopy cannot distinguish such fluctuations from those due to the rotation of the fluorophore, making the method susceptible to noise. The centroid-tracking method works only for off-axis rotation involves time-consuming, off-line image analysis. In general, it is difficult to observe rotation of a circular object at any scale when viewed along the axis of rotation unless the rotation of the object is eccentric to the axis of rotation and/or the rotating object has an asymmetric shape.